透過您的圖書館登入
IP:54.224.90.25
  • 學位論文

探討Irxl1基因在軟骨發育上的調控

Investigation of The Regulation of Irxl1 Gene in Cartilage Development

指導教授 : 潘惠錦

摘要


Irxl為一新穎基因,具有homeodomain,屬於homeobox gene中的 TALE次家族。因其homeodomain與IRX家族最為相似,因此命名為Irx- like 1(Irxl1)。斑馬魚胚胎發育過程中,Irxl1主要表現在腦部與咽喉鰓弓。利用anti-sense mopholino oligo (MO) 抑制內生性的Irxl1,會造成早期胚胎下顎軟骨的缺失。為了瞭解Irxl1基因的上游調控,我們分析了Irxl1基因上游(-1342/+249)序列,預測出數個Sox5的結合位。已知小鼠的Sox trio genes (Sox5、Sox6、Sox9)參與調控軟骨細胞分化的過程,而注射Sox5 MO至斑馬魚胚胎,五天後以Alcian blue染色觀察其下顎軟骨也有發育異常的情形,所以我們進一步探討Irxl1是否會藉由Sox5的調控影響軟骨的發育。藉由Dual luciferase報導基因分析,顯示Sox5或Sox5/Sox6會抑制Irxl1 promoter活性,而同時表達Sox5/Sox6/Sox9則會促進Irxl1的promoter活性,暗示Sox5和Sox trio genes可調控Irxl1並參與早期斑馬魚胚胎下顎軟骨的發育。另外我們也使用小鼠的軟骨前驅細胞ATDC5,藉由慢病毒轉染外源性shIrxl1基因來弱化Irxl1,探討Irxl1對於ATDC5細胞增生及分化過程產生的影響。ATDC5具有可誘導分化成軟骨及硬骨細胞的能力,我們使用此細胞株,觀察到穩定表達shIrxl1的細胞,其分化能力有下降的情形。Alcian blue與Alizarin red染色結果發現其細胞外基質的含量有減少,細胞分布的型態也改變。分析一些軟骨細胞分化的指標,顯示在弱化細胞株中ColI、ColII、ColX的表現是下降的,而分化過程中Irxl1與Sox5的表現互呈現消長的情形。我們觀察到Irxl1 弱化的細胞其貼附能力下降,利用定量PCR和Western分析,發現Integrin的表現量比控制組減少,其下游的蛋白p-FAK也減少,但p-ERK增加,同時MMP-13的表現也大幅上升。我們推測Irxl1與維持正常的細胞外基質有關,降低細胞貼附能力或許會影響到分化能力。

關鍵字

軟骨 分化

並列摘要


Iroquois homeobox-like 1 (Irxl1) is a novel member of the TALE superfamily of homeobox genes that is most-closely related to the Iroquois class. During zebrafish development, Irxl1 is expressed mainly in the brain and the first two pharyngeal arches. Antisense morpholino knockdown of Irxl1 results in defective pharyngeal cartilage in zebrafish larvae. Analysis of Irxl1 promoter reveals four putative Sox5 binding sites in the -1342/+249 region. Since the mouse Sox trio genes (Sox5, Sox6, Sox9) play a key role in regulating chondrocyte differentiation, we test the role of Sox5 in embryonic development by injecting antisense mopholino oligos of Sox5 into zebrafish embryonic. Alcian blue staining revealed defective pharyngeal cartilages in the morphant larvae of 5 days. Dual luciferase reporter assay of Irxl1 promoter revealed that overexpression of Sox5 or Sox5/Sox6 repressed that Irxl1 promoter activity, whereas the Sox trio genes together activated it. We also used ATDC5 cells to explore the role of Irxl1 on cell proliferation and differentiation. ATDC5 is, a chondrogenic cell line derived from a mouse teratocarcinoma that can be induced to differentiat to chondrocytes and osteocytes. We infected the ATDC5 cell with shIrxl1-lentivirus to knockdown endogenous Irxl1 expression and observed that Irxl1 knockdown resulted in delayed differentiation. Staining with alcian blue and alizarin red showed decreased extracellular matrix and altered cell morphology in knockdown cells. Real-time RT-PCR, analysis indicated that the expression of differentiation markers of chondrocyte ( ColI, ColII and ColX ) was decreased. The expression of Irxl1 and Sox5 was reciprocally related. In addition, the cell adhesion activity in knockdown cells is reduced. Both RNA and protein levels of integrin were decreased, as well as its downstream protein p-FAK. However, the level of p-ERK and MMP-13 were largely increased. The data suggest that Irxl1 may regulate the adhesion activity of ATDC5 cell and thus affect cell differentiation ability.

並列關鍵字

Irxl1 Sox5

參考文獻


Anderson, D.M., Beres, B.J., Wilson-Rawls, J., and Rawls, A. (2009). The homeobox gene Mohawk represses transcription by recruiting the sin3A/HDAC co-repressor complex. Developmental dynamics : an official publication of the American Association of Anatomists 238, 572-580.
Anderson, D.M., George, R., Noyes, M.B., Rowton, M., Liu, W., Jiang, R., Wolfe, S.A., Wilson-Rawls, J., and Rawls, A. (2012). Characterization of the DNA-binding properties of the Mohawk homeobox transcription factor. The Journal of biological chemistry 287, 35351-35359.
Challa, T.D., Rais, Y., and Ornan, E.M. (2010). Effect of adiponectin on ATDC5 proliferation, differentiation and signaling pathways. Molecular and cellular endocrinology 323, 282-291.
Chuang, H.N., Cheng, H.Y., Hsiao, K.M., Lin, C.W., Lin, M.L., and Pan, H. (2010). The zebrafish homeobox gene irxl1 is required for brain and pharyngeal arch morphogenesis. Developmental dynamics : an official publication of the American Association of Anatomists 239, 639-650.
Delgado-Olguin, P., Takeuchi, J.K., and Bruneau, B.G. (2006). Chromatin modification and remodeling in heart development. TheScientificWorldJournal 6, 1851-1861.

延伸閱讀